Protein tyrosine phosphatase receptor R and Z1 expression as independent prognostic indicators in oral squamous cell carcinoma

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1 ORIGINAL ARTICLE Protein tyrosine phosphatase receptor R and Z1 expression as independent prognostic indicators in oral squamous cell carcinoma Kamila Dus-Szachniewicz, MSc, 1 Marta Wozniak, MSc, 1 Kamil Nelke, MD, 2 El_zbieta Gamian, PhD, 1 Hanna Gerber, MD, PhD, 2 Piotr Ziołkowski, MD, PhD 1 * 1 Department of Pathology, Wrocław Medical University, Wrocław, Poland, 2 Department of Maxillo-Facial Surgery, Wrocław Medical University, Wrocław, Poland. Accepted 30 June 2014 Published online 27 January 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI /hed ABSTRACT: Background. The actions of tyrosine phosphorylation and dephosphorylation are controlled by tyrosine kinases and phosphatases. Although substantial previous data have revealed the role of several protein tyrosine phosphatases (PTPs) in various cancers, the function of protein tyrosine phosphatase receptor R (PTPRR) and protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1) proteins in oral cavity squamous cell carcinoma (SCC) has not been studied to date. Methods. The PTPRR and PTPRZ1 immunoreactivity in 67 formalin-fixed and paraffin-embedded oral cancer tissues at different stages were analyzed with the technique of immunohistochemistry (IHC). The presence of PTPRR in cancerous tissue was confirmed by Western blotting. Results. The occurrence of PTPRR and PTPRZ1 proteins in the cancer specimens was more frequent in lower grade tumors. In addition, the association between the immunoreactivity of both examined proteins and improved patients survival was detected. Moreover, the PTPRR expression was found to be related to the absence of synchronous lymph node involvement. Conclusion. The above results indicate that the PTPRR and PTPRZ1 protein expression should be monitored in oral cancer for patients prognostic stratification. VC 2015 Wiley Periodicals, Inc. Head Neck 37: , 2015 KEY WORDS: oral cavity squamous cell carcinoma (SSC), protein tyrosine phosphatases (PTPs), protein tyrosine phosphatase receptor R and Z1, immunohistochemistry, Western blot analysis INTRODUCTION Oral cavity squamous cell carcinoma (SCC) is the most frequent malignant tumor of the head and neck region and also one of the most common types of malignancy in Europe. In 2012, over 90,000 new cases of oral cancer had been reported, whereas over 40,000 patients with oral cancer died. 1 The incidence rates of this neoplasm are higher in men than in women and it is a major death-causing cancer in 50 to 60-year-old men. 2,3 The 5-year survival rate for oral cancer is still less than 50% and has not improved over the last few decades. 4 In recent years, a significant amount of research has been conducted in the study of etiology, molecular mechanisms, and genetic and environmental factors, such as alcohol and tobacco consumption, chronic inflammation, or viral infection. 5 7 The prevention of oral cancer and development of new therapeutic strategies requires the identification of the proteins crucial to the carcinogenesis process, to allow its etiopathology to be understood. Protein tyrosine phosphatases (PTPs) are large, heterogeneous groups of enzymes located in different parts of *Corresponding author: P. Ziołkowski, Department of Pathology, Wrocław Medical University, Marcinkowskiego 1, Wrocław, Poland. ziolkows@interia.pl the cellular compartments. Their effect is opposite to the action of protein tyrosine kinases these enzymes detach phosphate residues from other protein tyrosine enzyme, thereby deactivating them. 8 Moreover, some phosphatases (low molecular weight PTPs) remove phosphates from proteins resulting in the activation of kinases (eg, removal of phosphates from py527 Src turns in an increase of PTK activity). 9 It has been proved that abnormalities in protein tyrosine phosphorylation process lead to dysregulation of cellular signaling, which is strongly associated with carcinogenesis and tumor progression. 10 Protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1) is a member of the tyrosine phosphatase family. 11 This receptor is overexpressed in a wide number of tumors, including hepatocarcinoma, renal carcinoma, and glioblastoma. 11,12 PTP receptor type Z is also expressed in the mammalian central nervous system and has been suggested to influence regulation of oligodendrocyte survival and differentiation. It may play a role in multiple sclerosis and periventricular leukomalacia. 13 The PTPRR gene encodes the classical transmembrane PTP receptor type R. 14 Protein tyrosine phosphatase receptor type R (PTPRR) is a key negative regulator of the extracellular regulated kinase signaling pathway and its expression is regulated by androgen. There are authors who have suggested that some isoforms of the PTPRR 1816 HEAD & NECK DOI /HED DECEMBER 2015

2 PROTEIN TYROSINE PHOSPHATASE RECEPTOR R AND Z1 EXPRESSION IN ORAL CANCER gene may play a role in conferring a risk of major depressive disorder in female subjects. 15 The results of one study showed a statistically significant difference between promoter methylation in cancerous and healthy tissue, supporting the hypothesis that the protein tyrosine phosphatase receptor (PTPR) protein family has an important role in the etiology of colorectal carcinoma. 16 PTPRR gene silencing may thus represent a novel mechanism by which neoplastic colorectal cells evade tumor suppression. The assessment of PTPRR gene expression levels in different human tissues reveals preferential expression in the brain and lower gastrointestinal tract, thus, its downregulation might reasonably be expected to lead to the development of disease in these tissues. 17 In our previous study, we examined the level of expression of PTPRR and PTPRZ1 by immunochemistry in colorectal neoplasms and adjacent normal tissues. 18 The goal of the present study was twofold. First, to examine the PTPRR and PTPRZ1 occurrence in oral cancer tissue, and second, to ascertain whether there is any association of its expression with clinicopathological features. To our knowledge, the evaluation of the 2 above receptors in oral SCC has not been carried out to date. MATERIAL AND METHODS Patients The study was performed on tissue specimens from 67 male patients suffering from oral cavity SCC. Tissue samples were collected from 29 patients treated in the Clinical Hospital of Wrocław Medical University during the years 1998 to 2013, whereas a further 38 samples were obtained from patients diagnosed in Sokołowski Hospital in Wałbrzych during the same period of time. The patients ranged in age from 50 to 60 years and were selected according to the organ s localization of the cancer (the mucosa of the cheek, gingiva, tongue, hard or soft palate, tonsils, or oral part of throat) and tumor grade (G1, G2, G3). All patients underwent curative surgery and adequate radiotherapy or/and chemotherapy. None of the patients had received preoperative radiotherapy or chemotherapy. In our study, the 5-year survival, lymph node involvement, and presence of leukoplakia were evaluated for all cases. Ethics statement The investigation has been conducted in accordance with ethical standards, according to the Declaration of Helsinki and according to national and international guidelines, and has been approved by the authors institutional review board. The specimens were used upon approval by the local ethics committee. Immunohistochemistry All cases of oral cavity SCC have been histologically confirmed using hematoxylin-eosin staining of formalinfixed and paraffin-embedded specimens. All 5 mm thick sections were stained immunohistochemically using ABC method consisting on labeled streptavidin biotin reagents conjugated to horseradish peroxidase (LSAB1 System HRP; DAKO, Glostrup, Denmark). First, tissue slides TABLE 1. Staining intensity Immunoreactivity score calculation method. % of staining 0 5 lack of staining 0 5 0% to 10% 1 5 weak staining % to 25% 2 5 moderate staining % to 50% 3 5 strong staining % to 75% 4 5 >75% The final score was calculated by multiplying staining intensity value (integral score 0 3) by percentage of stained cells (integral score 0 4). The resulting immunoreactivity score ranged from 0 to 12. were dried 12 hours at 60 C, then deparaffinized in xylene, rehydrated in a series of graded alcohols, and antigen-retrieved by microwaving for 5 minutes in citrate buffer, ph Peroxidase activity was blocked for 10 minutes with peroxidase blocking reagent and then sections were incubated for 30 minutes with 10% normal goat serum at room temperature to reduce nonspecific reaction. Two primary antibodies were purchased from Atlas Antibodies (Stockholm, AB Sweden), and used in the study: HPA rabbit polyclonal antibody PTPRZ1, and HPA rabbit polyclonal antibody PTPRR. Sections were incubated with the above antibodies (dilution 1:150) and stored overnight at 4 C. Next, the slides were incubated for 15 minutes with each of the following 2 reagents: biotinylated link antibody and streptavidin conjugated to horseradish peroxidase (HRP) with rinsing twice with phosphate buffered saline (PBS) between and after them and stained with 3,3 0 -diaminobenzidine (DAB) in chromogen solution. Finally, sections were counterstained with Mayer s hematoxylin and then dehydrated in graded alcohols, cleared in xylene, and mounted with xylene-based mounting medium. Negative control was obtained by omitting the first, polyclonal antibody, whereas the positive controls were achieved by using the samples of colorectal carcinoma known to express both PTPRR and PTPRZ1. Immunohistochemistry evaluation Immunostaining of tumor sections for PTPRR and PTPRZ1 was reported according to the following standards (Table 1). For each specimen, the immunoreactivity score (IRS) was calculated by multiplying staining intensity value (integral score, 0 3) by percentage of stained cells (integral score, 0 4), as described in Table 1. The resulting IRS ranged from 0 to 12. The percentage of stained cells was calculated from 5 neighboring fields (3100) in the center of lesions and the total number of stained cells was then divided by 5. For tumor grade immunoreactivity, the results were considered as negative (0), weakly positive (1 4), positive (5 8), and strongly positive (9 12). For other clinicopathological features, the IRS was categorized as negative (0 3) and positive (4 12). The slides were evaluated by 2 independent pathologists in a blind manner using the light microscope (Leica, Wetzlar, Germany) at 3100 magnification. The scores then were compared between pathologists and their validation was in agreement in 85.3%. A final 100% consensus was assigned after the second evaluation of discordant cases by pathologist. HEAD & NECK DOI /HED DECEMBER

3 DUS SZACHNIEWICZ ET AL. FIGURE 1. Protein tyrosine phosphatase receptor R (PTPRR) and Z1 (PTPRZ1) expression in oral cavity squamous cell carcinoma analyzed by immunohistochemistry. Cytoplasmic pattern of immunohistochemical staining was observed for both analyzed proteins. (A) Strong immunostaining of PTPRR in well-differentiated tumor. (B) Strong immunostaining of PTPRZ1 in well-differentiated tumor. (C) Weak immunostaining of PTPRR in poorly differentiated carcinoma. (D) Negative staining of PTPRZ1 in poorly differentiated carcinoma. Antibodies were detected by diaminobenzidine (DAB) method, the counterstaining of tissue was done with hematoxylin (original magnifications 3200). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Western blot analysis For the Western blot, formalin-fixed and paraffinembedded tumor blocks were used. First, the tumor blocks were cut into ten 20-um sections and put on microscope slides. Next, the pathologist marked the cancerous parts of the sections in accordance with the image visible in the light microscope and the proper parts of each section were macrodissected. Collected tissues were deparaffinized in 3 changes of xylene in a heating block with agitation, 10 minutes each, clear in 3 changes of absolute alcohol and dry in concentrator (Eppendorf, Hamburg, Germany). Then, the samples were homogenized in a lysis buffer (0.1 M Tris-HCL, ph 8.0, 0.1 M DL-dithiothreitol, 4% sodium dodecyl sulfate) in 99 Cin a heating block with agitation (600 rpm) for 1 hour. Next, the crude extracts were clarified by centrifugation at 16,000 3 g at 18 C for 10 minutes. The protein concentration in supernatant was measured at 280 nm using a NanoDrop 2000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose (Amersham Hybond; GE Healthcare Bio-sciences AB, Uppsala, Sweden) by NuPage System recommended by Invitrogen (Life Technologies, Carlsbad, CA). After washing in PBS, incubation was done with 2.5% glutaraldehyde (Sigma Aldrich, Seelze, Germany) and then with 5% goat serum (Sigma Aldrich), the membrane was probed with primary antibody against PTPRR (dilution 1:250) at 4 C. The anti-b-actin polyclonal antibody was purchased from Abcam (Cat. No. ab8227; Abcam, Cambridge, UK). The following day, after washing with PBS-Tween 0.1%, blots were incubated with the appropriate HRP-conjugated secondary antibody (Abcam, Cambridge, UK) and the bound antibodies were visualized using DAB Enhanced Liquid Substrate System for immunohistochemistry (IHC; Sigma Aldrich). Documentation of bands was performed using Molecular Imager Gel Doc TMXR1 (BioRad, Hercules, CA). Statistical analysis Statistical analysis was performed by STATISTICA v.10.0 (StatSoft, Krakow, Poland). The association between PTPRR and PTPRZ1 immunoreactivity and clinicopathological parameters were analyzed by Pearson s chi-square test or Fisher s exact test. Multiple comparison of PTPRR and PTPRZ1 protein expression among G1, G2, and G3 tumor grade were analyzed by Kruskal Wallis test. The relationship between PTPRR and PTPRZ1 staining was analyzed by Fisher s exact test. Odds ratio (OR) were calculated for the data presented as 4-field table according to the formula: OR 5 (TP/FN)/(FP/TN) where TP, FN, FP, and TN are the number of true-positives, false HEAD & NECK DOI /HED DECEMBER 2015

4 PROTEIN TYROSINE PHOSPHATASE RECEPTOR R AND Z1 EXPRESSION IN ORAL CANCER TABLE 2. Immunoreactivity score for protein tyrosine phosphatase receptor R and Z1 in oral cavity squamous cell carcinoma tissue by tumor grade. Immunoreactivity score for PTPRR Immunoreactivity score IRS for PTPRZ1 Grade G1 No. of patients, 21 0 (0%) 3 (14.3%) 7 (33.3%) 11 (52.4%) 9 (42.8%) 6 (28.6%) 5 (23.8%) 1 (4.8%) G2 No. of patients, 33 4 (12.1%) 10 (30.3%) 9 (27.3%) 10 (30.3%) 18 (54.5%) 9 (27.3%) 5 (15.2%) 1 (3%) G3 No. of patients, 13 9 (69.2%) 2 (15.4%) 2(15.4%) 0 (0%) 12 (92.3%) 1 (7.7%) 0 0 Abbreviations: PTPRR, protein tyrosine phosphatase receptor R; IRS, immunoreactivity score; PTPRZ1, protein tyrosine phosphatase, receptor-type, Z polypeptide 1. The IRS evaluation was defined in Materials and Methods. negatives, false-positives, and true-negatives, respectively. The SE of the log OR is approximately: SE(log OR) 5 (1/TP 11/FN 11/FP 1 TN). From this, an approximate 95% confidence interval (CI) can be calculated for the log odds ratio: log(or) * SE(log OR). An OR with its 95% CI was measured as the effect size estimate. Any p <.05 was considered as a statistically significant in all analyses. RESULTS In order to study the PTPRR and PTPRZ1 protein expression in oral cancer specimens, the histological slides were stained against PTPRR and PTPRZ1 with relevant antibodies. For both proteins, the IHC staining was cytoplasmic with no nuclear reaction (Figure 1). As demonstrated in Table 2, for PTPRR 80.6% (54 of 67) tissues were positive (IRS ), whereas for PTPRZ1 there were only 41.8% (28 of 67). The negative control, in which the blocking solution was used instead of primary antibodies, showed no signal. Association with clinicopathological features In this research, the immunoreactivity of PTPRR and PTPRZ1 with regard to the patients clinicopathological characteristics was analyzed. For PTPRR protein, its expression was significantly related to the lymph node metastasis status and 5-year survival, whereas no association was observed with tumor localization, presence of leukoplakia, and cigarette smoking. The overexpression of PTPRZ1 was found to be strongly associated with patient survival. Additionally, the significant difference in PTPRR and PTPRZ1 protein expression was observed among G1, G2, and G3 tumors. Tumor grade With respect to the tumor grade, in well-differentiated tumors (G1), there were no PTPRR-negative cases observed, whereas 52.4% (11 of 21) of all specimens showed high immunoreactivity (Table 2). A total of 12.1% (4 of 33) of moderately differentiated tumors (G2) were negative, whereas the percentage of patients in other IRS groups was around 30%. In contrast, 69.2% (9 of 13) of poorly differentiated tumors (G3) were negative and 15.4% (2 of 13) showed weak (IRS 5 1 4)ormoderate(IRS55 8) patterns of immunoreactivity. In G3 tumors, strong immunostaining (IRS ) was not observed. To summarize, significantly higher PTPRR expression was observed in G1 (p <.001) and G2 cases (p 5.003) compared to G3. FIGURE 2. Multiple comparison of protein tyrosine phosphatase receptor R (PTPRR; left) and Z1 (PTPRZ1; right) immunoreactivity score (IRS) among G1, G2, and G3 tumor grades in 67 patients with oral cancer. For PTPRR protein expression, the significance difference between G1 (p <.001) and G2 cases (p 5.003) according to G3 was assessed. For PTPRZ1 immunoreactivity, there were significant differences between G1 and G3 tumors (p <.05). The Kruskal Wallis test was used to compare results between groups. A p <.05 was considered statistically significant. HEAD & NECK DOI /HED DECEMBER

5 DUS SZACHNIEWICZ ET AL. TABLE 3. Association between protein tyrosine phosphatase receptor R and Z1 protein expression and patients clinicopathological features. PTPRR PTPRZ1 Characteristics No. of cases Negative Positive p value* Negative Positive p value* Lymph node involvement No 54 (80.6%) 6 (11.1%) 48 (89.9%) 29 (53.7%) 25 (46.3%) Yes 13 (19.4%) 7 (53.8%) 6 (46.2%) 10 (76.9%) 3 (23.1%) Tumor localization Cheek 15 (22.4%) 4 (26.7%) 11 (73.3%) 8 (53.3%) 7 (46.7%) Tongue 18 (26.9%) 2 (11.1%) 16 (88.9%) 9 (50.0%) 9 (50.0%) Tonsils 11 (16.4%) 2 (18.2%) 9 (81.8%) 5 (45.5%) 6 (54.5%) Palate 11 (16.4%) 3 (27.3%) 8 (72.7%) 8 (72.7%) 3 (27.3%) Gingiva 12 (17.9%) 2 (16.7%) 10 (83.3%) 9 (75.0%) 3 (25.0%) Smokers Yes 10 (14.9%) 1 (10.0%) 9 (90.0%) 6 (60.0%) 4 (40.0%) No 32 (47.8%) 9 (28.1%) 23 (71.9%) 19 (59.4%) 13 (40.6%) No data 25 (37.3%) 3 (12.0%) 22 (88.0%) 14 (56.0%) 11 (44.0%) Leukoplakia Yes 25 (37.3%) 3 (12.0%) 22 (88.0%) 14 (12.0%) 11 (88.0%) No 42 (62.7%) 10 (23.8%) 32 (76.2%) 25 (23.8%) 17 (76.2%) Survival <.001 <.001 >5 y 45 (67.2%) 11 (24.4%) 34 (75.6%) 20 (44.4%) 25 (55.6%) <5 y 22 (32.8%) 12 (54.5%) 10 (45.5%) 19 (86.4%) 3 (13.6%) Abbreviations: PTPRR, protein tyrosine phosphatase receptor R; PTPRZ1, protein tyrosine phosphatase, receptor-type, Z polypeptide 1. * Pearson s chi-square test. Odds ratio for PTPRR: OR ; 95% confidence interval (CI) Odds ratio for PTPRZ1, Fisher s exact test: OR ; 95% CI The p value in bold indicate statistical significance. The p values were calculated by Pearson s chi-square test or and Fisher s exact test when appropriate. There was no significant difference in PTPRR protein level between G1 and G2 tumors (Figure 2). The expression of PTPRZ1 protein by IHC was negative in 42.8% (9 of 21), 54.5% (18 of 33), and 92.3% (12 of 13) for G1, G2, and G3 tumors, respectively (Table 2). For G1 and G2 specimens, the majority of PTPRZ1-positive cases showed weak immunoreactivity (IRS 5 1 4), whereas strong immunostaining (IRS ) was observed in 4.8% (1 of 21) and 3% (1 of 33), respectively. For G3 tumors, there was no moderate and strong expression of PTPRZ1 detected. The multiple comparison of PTPRZ1 IRS among G1, G2, and G3 tumors proved that significantly higher PTPRZ1 protein expression was observed in G1 tumors compared to G3 (p <.05), whereas no significant differences were detected between other groups. Lymph node involvement In this study, PTPRR protein was positive (IRS ) in 89.9% (48 of 54) of specimens without lymph node involvement, whereas in the examined cases with lymph node involvement, the positive expression was only 46.2% (6 of 13; p <.01; Table 3). In contrast, no statistically important association was detected between lymph node involvement and PTPRZ1 immunoreactivity. Survival Analysis of Pearson s chi-square test for PTPR and Fisher s exact test for PTPRZ1 demonstrated a significant impact of both examined receptors on patients survival (Table 3). The results obtained in this study established that 75.6% (34 of 45) of patients with oral cancer with positive PTPRR (IRS ) showed an overall survival rate of >5 years. In contrast, among patients who had survival rate <5 years, 86.4% (19 of 22) of cases were PTPRZ1-negative (IRS 5 0 3). Additionally, OR calculation showed that patients with positive PTPRR had a >5-year survival rate nearly 4 times higher in comparison with patients with negative PTPRR staining. The OR for positive PTPRZ1 showed almost 8 times lower risk of death within 5 years. Other clinicopathological characteristics As demonstrated in Table 3, there was no significant association observed between PTPRR and PTPRZ1 protein expression and certain features of oral cancer, including tumor localization, chronic cigarette smoking, and the presence of leukoplakia. Association between protein tyrosine phosphatase receptor R and protein tyrosine phosphatase, receptor-type, Z polypeptide 1 immunoreactivity Fisher s exact tests were used to assess the association between PTPRR and PTPRZ1 staining. This study established that the expression of PTPRR protein is associated with the PTPRZ1 immunoreactivity (Table 4). The OR showed that PTPRR-positive status was almost 12 times more frequent when PTPRZ1 was positive than PTPRZ1 was negative Western blot analysis The PTPRR protein expression in formalin-fixed and paraffin-embedded tumor samples was confirmed by the Western blot technique. The strong PTPRR expression 1820 HEAD & NECK DOI /HED DECEMBER 2015

6 PROTEIN TYROSINE PHOSPHATASE RECEPTOR R AND Z1 EXPRESSION IN ORAL CANCER TABLE 4. Fisher s exact test of association between protein tyrosine phosphatase receptor R and Z1 expression in patients with oral cancer. PTPR positive PTPR negative p value PTPRZ1 positive 27 (40.3%) 1 (1.5%).005 PTPRZ1 negative 27 (40.3%) 12 (17.9%) Abbreviations: PTPR, protein tyrosine phosphatase receptor R; PTPRZ1, protein tyrosine phosphatase, receptor-type, Z polypeptide 1. Odds ratio and 95% confidence interval was found in 2 samples from G1 patients with oral cancer. In contrast, PTPRR was not detected in any of the G3 samples. The results of the Western blot analysis are presented in Figure 3. The relevant Western blot analysis of PTPRZ1 was not performed because, according to the manufacturer s data, the available antibody is not recommended for such analysis. DISCUSSION It is well known that protein phosphorylation plays an important role in cell signaling and in the regulation of cellular functions. 17,19 The levels of cellular phosphorylation are reversibly controlled by protein tyrosine kinases and PTPs. 20,21 It has been thought that tyrosine phosphatases act as tumor suppressors, 14,15 however, this statement has been recently questioned as the latest data suggests that dephosphorylation events induced by PTPs stimulate the growth of some tumors. PTP genes could then have a dual role as both tumor suppressors and oncogenes; consequently its function in the development of cancer still remains unclear. To date, the role of PTPRR and PTPRZ1 proteins in cancer has not been studied extensively over the past years, the association between PTPRR and human malignancy was reported only for cervical and colon cancer. 17,24 Many genetic studies are based on the hypothesis that PTPR genes are downregulated in cancers. However, our previous and present findings are changing the current point of view we have shown the overexpression of 2 PTPRs, PTPRZ1 and PTPRR in malignant tissues. 18 This study is the first investigation of the expression of both proteins in oral cancer specimens using IHC and Western blot analysis. Our main purpose of this study was to establish whether there is any association between PTPRR and PTPRZ1 protein expression in IHC and patients clinicopathological features. Recently, Thomas et al 25 confirmed a strong association between histologic grade and survival in patients with oral cancer. They found that well-differentiated tumors are associated with a better overall survival rate. In our study, we analyzed the differences in PTPRR and PTPRZ1 immunoexpression among G1, G2, and G3 tumor grade. We established that G1 and G2 tumors were PTPRR-positive in 100% and 87.9% cases, respectively, whereas almost 70% of G3 tumors were PTPRR negative. At the same time, patients with positive PTPRR protein expression had 4 times higher survival over 5 years compared with PTPRR-negative patients. For PTPRZ1 protein, we found that it was significantly higher expressed in G1 tumors compared to G3. Moreover, PTPRZ1- positive expression in oral cancer tissue was found to be associated with PTPRR-positive staining. In order to see whether PTPRR differential expression within the histologic tumor grade is a common feature of human cancers, additional studies on this protein expression in different types of human malignancies may be required. The prognosis for patients with oral cavity SCC depends, among other factors, on the presence of lymph node metastases and distant metastases. In the present study, we found a significant relationship between PTPRR immunoreactivity and lymph involvement status. The tumor specimens from 89.9% of patients without lymph node metastasis demonstrated positive PTPRR protein expression. These data allow us to conclude with the hypothesis that PTPRR acts as a tumor suppressor and inhibits the cancer invasion and metastasis. These findings remain in concordance with the investigation of Su at al 24 who has confirmed the same PTPRR function in CaSki and HeLa3rd cells. Nonetheless, further analysis of PTPRR protein expression in oral cancer lesions depending on lymph node involvement status is needed in future studies to explain the exact function of PTPRR in cancer progression. Currently, patients with early stage tumors are treated with surgery or radiotherapy and generally have a satisfactory prognosis. 26 The standard treatment for advanced oral cancers is surgery associated with chemotherapy and/ or adjuvant radiotherapy. In some cases, the combined strategies are more efficient than surgery alone. However, the fact remains that chemotherapy is still very costly, and increases the risk of serious side effects. Therefore, it is crucial to classify the patients in order to deliver the most effective treatment. For this purpose, new prognostic and predictive biomarkers are required. We suggest that the validation of PTPRR and PTPRZ1 protein expression in patients with oral cancer may provide important FIGURE 3. Western blot analysis of protein tyrosine phosphatase receptor R (PTPRR) protein (22 kda) in formalin-fixed paraffinembedded tissue from 4 patients with oral cancer (I IV). I and II represent the presence of PTPRR in G1 tumors, whereas III and IV show the lack of PTPRR in examined G3 specimens. Antibodies against PTPRR was diluted 1:250, for internal control against b-actin (42 kda) in dilution 1:1000. HEAD & NECK DOI /HED DECEMBER

7 DUS SZACHNIEWICZ ET AL. information on the likely course of the cancer disease and may be useful in the selection of the best tailored therapy. The limitations of this study include the small number of patients enrolled. Additionally, it is inappropriate to determine from obtained data whether the expressions of PTPRR and PTPRZ1 proteins are independent markers of survival, because patients had undergone the differential postoperative treatment. Nonetheless, PTPRR is a prognostic marker of lymph node metastasis that determines the effectiveness of treatment and may have a significant impact on patients survival. CONCLUSION In conclusion, our results indicate that PTPRR protein may play a role in tumor suppression. We claim that expression of PTPRR in cancerous tissues prevents the development of the well and moderately differentiated tumors into more aggressive, poorly differentiated tumors. Furthermore, the PTPRR and PTPRZ1 status gains a prognostic value for patients with oral cancer. We can believe that PTPRR protein expression in the absence of PTPRZ1 may help to distinguish patients with a low risk of developing lymph node metastasis and could be useful marker in the decision to perform additional lymph node excision in patients affected by oral cavity SCC. REFERENCES 1. European Cancer Observatory. Available at: Accessed October 1, Edwards BK, Brown ML, Wingo PA, et al. Annual report to the nation on the status of cancer, , featuring population-based trends in cancer treatment. J Natl Cancer Inst 2005;97: Barnes L, Eveson JW, Reichart P, Sidransky D. Pathology and Genetics of Head and Neck Tumours, third edition. World Health Organization Classification of Tumors, volume 9. Lyon, France: IARC Press; Pasini FS, Maistro S, Snitcovsky I, et al. Four-gene expression model predictive of lymph node metastases in oral squamous cell carcinoma. 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